Inhibitor Of ATP-citrate Lyase Research Articles

The source of acetyl coenzyme A for acetylcholine synthesis in the perfused rat phrenic nerve-hemidiaphragm.

Acetylcholine release by the phrenic nerve was measured in the isolated, perfused rat hemidiaphragm. In controls, the rate of release of acetylcholine increases with time; however, when (--)-hydroxycitrate, an inhibitor of ATP-citrate lyase, is added to the perfusate, the release of acetylcholine stabilizes at a level 40% below the final control value. In this preparation, the capacity of the citrate cleavage pathway to transfer acetyl coenzyme A from the nerve cell mitochondria to the cytosol increases with time; this is not the case for other transport processes.

The effects of specific lipogenic substrates and metabolic inhibitors on de Novo fatty acid synthesis in isolated hepatocytes from chow-fed female rats

The effects of glucose (10 m m), glycerol (3 m m), and lactate/pyruvate (10 m m) on the incorporation of 3H from 3H 2O into fatty acids were studied in isolated hepatocytes prepared from chow-fed female rats. Lactate/pyruvate markedly increased lipogenic rates, while glucose and glycerol did not significantly affect rates of lipogenesis. In cells incubated with lactate/pyruvate plus glycerol, the increase in 3H incorporation was greater than observed with lactate/pyruvate alone. In hepatocytes isolated from 24-h starved rats, lactate/pyruvate again increased de novo fatty acid synthesis to a greater extent than either glucose or glycerol. Glycerol significantly increased lipogenesis compared to the endogenous rates and when incubated with lactate/pyruvate produced an increase above lactate/pyruvate alone. (−)-Hydroxycitrate, a potent inhibitor of ATP-citrate lyase (EC 4.1.3.8), and agaric acid, an inhibitor of tricarboxylate anion translocation, were studied in hepatocytes to determine their effects on lipogenesis by measuring 3H 2O, [1- 14C]acetate, and [2- 14C]lactate incorporation into fatty acids. 3H incorporation into fatty acids was markedly inhibited by both inhibitors with agaric acid (60 μ m) producing the greater inhibition. (−)-Hydroxycitrate (2 m m) increased acetate incorporation into fatty acids from [1- 14C]acetate and agaric acid produced a strong inhibitory effect. Combined effects of (−)-hydroxycitrate and agaric acid on lipogenesis from [1- 14C]acetate showed an inhibitory response to a lesser extent than with agaric acid alone. With substrate concentrations of acetate present, there was no significant increase in rates of lipogenesis from [1- 14C]acetate and the increase previously observed with (−)-hydroxycitrate alone was minimized. Agaric acid significantly inhibited fatty acid synthesis from acetate in the presence of exogenous substrate, but the effect was decreased in comparison to rates with only endogenous substrate present. With [2- 14C]lactate as the lipogenic precursor, agaric acid and (−)-hydroxycitrate strongly inhibited fatty acid synthesis. However, agaric acid despite its lower concentration (60 μ m vs 2 m m) was twice as effective as (−)-hydroxycitrate. A similar pattern was observed when substrate concentrations of lactate/pyruvate (10 m m) were added to the incubations. When (−)-hydroxycitrate and agaric acid were simultaneously incubated in the presence of endogenous substrate, there was an additive effect of the inhibitors on decreasing fatty acid synthesis. Results are discussed in relation to the origin of substrate for hepatic lipogenesis and whether specific metabolites increase lipogenic rates.

Lipogenesis in the brain of suckling rats. Studies on the mechansim of mitochondrial-cytosolic carbon transfer.

1. Studies on the incorporation of [3-(14)C]pyruvate and d-3-hydroxy[3-(14)C]butyrate into the brain lipid fraction by brain homogenates of the suckling (7-day-old) rat have been carried out. 2. Whereas approximately twice as much CO(2) was evolved from pyruvate compared with 3-hydroxybutyrate metabolism, similar amounts of the radioactivity of these two precursors were incorporated into the lipid fraction. Furthermore, in both cases the incorporation into lipid was almost tripled when glucose (10mm) or NADPH (2.5mm) was added to the incubation media. 3. If 5mm-(-)-hydroxycitrate, an ATP-citrate lyase inhibitor, was added to the incubation the incorporation of carbon from pyruvate was inhibited to 39% of the control and from 3-hydroxybutyrate to 73% of the control, whereas CO(2) production from both precursors was not affected. 4. The incorporation from pyruvate or 3-hydroxybutyrate into lipids was not affected by the presence of 10mm-glutamate in the medium (to encourage N-acetylaspartate production). However, incorporation from pyruvate was inhibited by 21% in the presence of 5mm-amino-oxyacetate (a transaminase inhibitor) and by 83% in the presence of both hydroxycitrate (5mm) and amino-oxyacetate. 5. Incorporation from 3-hydroxybutyrate into brain lipids was inhibited by 20% by amino-oxyacetate alone, but by 55% in the presence of both hydroxycitrate and amino-oxyacetate. 6. It is concluded that the mechanism of carbon transfer from pyruvate into lipids across the mitochondrial membrane in the suckling rat brain is mainly via citrate and N-acetylaspartate. 3-Hydroxybutyrate, in addition to using these routes, may also be incorporated via acetoacetate formation and transport to the cytosol.

Metabolic regulation as a control for lipid disorders. II. Influence of (——)-hydroxycitrate on genetically and experimentally induced hypertriglyceridemia in the rat

These studies were designed to determine whether genetically and experimentally induced hypertriglyceridemia were correlated with hyperlipogenesis, and whether inhibiting fatty acid synthesis would reduce serum triglyceride levels. Hypertrigylceridemia, resulting from genetic obesity in Zucker rats and fructose feeding or Triton administration to Charles River rats, was examined in relation to in vivo rates of heatic fatty acid synthesis, and the influence of (--)-hydroxycitrate (a potent competitive inhibitor of ATP citrate lyase) on serum triglyceride levels and lipogenesis was determined. Zucker obese rats demonstrated significantly increased rates of fatty acid synthesis and levels of serum triglycerides compared to their lean litter mates; lipogenic rates and circulating triglycerides were reduced markedly by the oral administration of (--)-hydroxycitrate. Fructose administered in the diet or drinking water induced a hypertriglyceridemia which was associated with a marked increase in hepatic lipogenesis, and (--)-hydroxycitrate reduced significantly both parameters. In contrast to the significant role that increased rates of lipogenesis apparently played in the development of hypertriglyceridemia in the Zucker rat and fructose-fed rat, Triton given intravenously produced a marked rise in serum triglycerides which could not be accounted for, to an appreciate extent, by increased rates of fatty acid synthesis. (--)-Hydroxycitrate reduced serum triglyceride levels and hepatic lipogenic rates equivalently in the Triton-treated and nontreated rats.

Pulmonary fatty acid synthesis. I. Mitochondrial acetyl transfer by rat lung in vitro.

Incorporation of tritiated water into fatty acids by rat adipose tissue and lung tissue slices incubated with 5 mM glucose indicated a level of fatty acid synthesis in rat lung approximately 15% that observed in adipose tissue in vitro. (-)-Hydroxycitrate, and inhibitor of ATP citrate lyase, markedly reduced tritiated water incorporation into fatty acids by lung tissue slices. The effects of (-)-hydroxycitrate and n-butymalonate on the incorporation of 14C-labeled glucose, pyruvate, acetate, and citrate suggested that citrate is a major acetyl carrier for de novo fatty acid synthesis in lung tissue. Alternative mechanisms to citrate as an acetyl carrier were also considered. Lung mitochondrial preparations formed significant levels of acetylcarnitine in the presence of pyruvate and carnitine. However, the effect of carnitine on the incorporation of 14C-labeled glucose, pyruvate, acetate, and citrate into fatty acids by lung tissue slices indicated that acetylcarnitine may not be a significant acetyl carrier for fatty acid synthesis but may serve as an acetyl "buffer" in the control of mitochondrial acetyl-CoA levels. Additionally, it appears unlikely that either acetylaspartate or acetoacetate are of major importance in acetyl transfer in lung tissue.

Lipogenesis from ketone bodies in rat brain. Evidence for conversion of acetoacetate into acetyl-coenzyme A in the cytosol

The metabolism of acetoacetate via a proposed cytosolic pathway in brain of 1-week-old rats was investigated. (-)-Hydroxycitrate, an inhibitor of ATP citrate lyase, markedly inhibited the incorporation of carbon from labelled glucose and 3-hydroxybutyrate into cerebral lipids, but had no effect on the incorporation of labelled acetate and acetoacetate into brain lipids. Similarly, n-butylmalonate and benzene-1,2,3-tricarboxylate inhibited the incorporation of labelled 3-hydroxybutyrate but not of acetoacetate into cerebral lipids. These inhibitors had no effect on the oxidation to 14CO2 of the labelled substrates used. (-)-Hydroxycitrate decreased the incorporation of 3H from 3H2O into cerebral lipids by slices metabolizing either glucose or 3-hydroxybutyrate, but not in the presence of acetoacetate. (-)-Hydroxycitrate also differentially inhibited the incorporation of [2-14C]-leucine and [U-14C]leucine into cerebral lipids. The data show that, although the acetyl moiety of acetyl-CoA generated in brain mitochondria is largely translocated as citrate from these organelles to the cytosol, a cytosolic pathway exists by which acetoacetate is converted directly into acetyl-COA in this cellular compartment.

Effect of halofenate and clofibrate on lipid synthesis in rat adipocytes

The free acids of the plasma lipid-lowering agents, halofenate and clofibrate inhibited the incorporation of radioactive glucose and pyruvate into fatty acids of isolated adipocytes prepared from rat epididymal fat pads. The concentration which inhibited fatty acid synthesis was dependent on the bovine serum albumin concentration in the incubation. The 50% inhibitory concentration of the free acid of halofenate in 1%, 2% and 4% albumin was 0.9 mM 2.3 mM and 4.4 mM, respectively. The potency of clofibrate was also lowered by increasing the albumin concentration. These compounds inhibited the uptake of both [ 14C]glucose and [ 14C]pyruvate to the same degree as the incorporation of these substrates into fatty acids. However, the drugs either had no effect on, or stimulated the uptake of palmitate by the cells. Leucine accumulation by the adipocytes was unaffected by halofenate (free acid) and inhibited by clofibrate (free acid). A comparison of these agents with (−)-hydroxycitrate, kynurenate and cerulenin (inhibitors of ATP-citrate lyase, acetyl CoA carboxylase and fatty acid synthetase, respectively) on the oxidation of pyruvate suggested that they inhibited pyruvate metabolism at or near the enzyme, pyruvate dehydrogenase.